Thermal Proteome Profiling Journal Club - 26.01.2021 Emina Lemeš The Holy Grail: Identifying Effective and Safe Therapies • Small molecule drugs • Main challenge: identification of molecular targets underlying drug therapeutic effects • and/or adverse side effects Global Investigation of Protein-ligand Interactions • Affinity-based target identification techniques • Drug affinity responsive target stability (DARTS) • Limited proteolysis mass spectrometry • Thermal proteome profiling (TPP)t Global Investigation of Protein-ligand Interactions • Affinity-based target identification techniques • Limited by necessity to modify each drug individually (without losing bioactivity) • Drug affinity responsive target stability (DARTS) • Limited proteolysis mass spectrometry • Thermal proteome profiling (TPP) Global Investigation of Protein-ligand Interactions • Affinity-based target identification techniques • Drug affinity responsive target stability (DARTS) • Drugs binding to target = stabilization • Reducing protease sensitivity by masking recognition sites • Limited proteolysis mass spectrometry • Thermal proteome profiling (TPP) (Lomenick et al 2009, PNAS) Mass Spectrometry Global Investigation of Protein-ligand Interactions • Affinity-based target identification techniques • Drug affinity responsive target stability (DARTS) • Limited proteolysis mass spectrometry • Thermal proteome profiling (TPP) Thermal Proteome Profiling (TPP) - Paper 1 Science • 3 OCTOBER 2014 • VOL 346 ISSUE 6205 1255784-1 Thermal Proteome Profiling – Workflow Extraction: PBS Reporter ion Cells cultured Each condition Digestion: Briefliy heated intensities under -> cells divided trypsin to a different Analyzed by used to fit a differential into 10 temperature Labeling: LC-MS/MS melting curve conditions aliquots TMT10 isotope for each tag protein Around intrinsic melting temperature proteins denature and aggregate Gradual disappearance from PBS- extracted samples with rising temperaure ! Only for soluble proteome fraction Differences Between Cells and Extract • K562 cells (human, suspension, chronic myeloid leukemia) • Quantitative thermal stability data for 5299 1st description of a melting proteome «meltome» proteins across 10 different temperatures • 2 exp setting: heating of intact cells or cell extracts • In both: weak but significant anticorr of thermal stability with molecular weight -> smaller proteins tend to be more stable Differences Between Cells and Extract • 3204 proteins robustly quantified in both cells and cell extracts • Hierarchical cluster analysis of the temperature-dependent relative protein concentrations Observed: • Group 1) Increased stability at 50°C and pronounced decrease at 56°C • Group 2) Increase at 63°C No conc increases by heating • Due to initial solubilization followed by aggregation at higher temp • GO analysis Fold-change (rel. to lowest temp 37°/40°C cells /extract resp) Gene Ontology Analyis in Intact Cells: • Proteins in these Clusters are released from: • or large protein assemblies (ribosomes) • disintegrating organelles (eg mitochondria) Melting Point Determination For Each Protein Passing quality control criteria Greater thermal stability (higher Tm values) Greater Thermal Stability in Cell Extract Compared to Intact Cells Average of 2.7°C higher Tm values in cell extract compared with intact cells. Disruption of the cellular context 2 2 2 R = 0.89 R = 0.30 heterogeneously affects protein stability R = 0.93 - due to lower protein concentration? Cellular context should have a stabilizing effect because -> molecular crowding. But results are contrary. Prev studies showed: phosphoglycerate kinase more stable in intact cell. Hypothesis: Phosphoglycerate kinase Then extraction might stabilization could also be explained by cause dissociation and binding of endogenous co-substrate ATP show lower Tm Greater Thermal Stability in Cell Extract Compared to Intact Cells 2 2 Cellular context should have a stabilizing R2 = 0.93 R = 0.89 R = 0.30 effect because -> molecular crowding. But results are contrary. Prev studies showed: phosphoglycerate kinase more stable in intact cell. Hypothesis: Phosphoglycerate kinase stabilization could also be explained by binding of endogenous co-substrate ATP Then extraction might cause dissociation and show lower Tm Or Does Cellular Context Have an Impact on Thermal Stability? – ATP-Binders Analysis of Tm values of 440 annotated ATP binders -> Tm higher Tm (increased stability) in intact cells when compared with all other proteins ATP-Binders Are Stabilized in Cell Extract m With the addition of MgATP Tm Analysis of Tm values of 440 annotated ATP binders -> higher Tm in intact cells compared to cell extract K562 cell extract supplemented with Tm MgATP (2mM) -> only ATP binders show trend towards higher Tm Another Experiment with DNA – binders (p53 and cognate DNA) -> ATP binders (2 replicate experiments) DNA-Binders Are Stabilized Upon Ligand Binding Tm When supplemented with MgATP -> only ATP binders show trend towards higher Tm Another Experiment with DNA – binders (p53 and cognate DNA) -> same results (Binding deficient mutant) Proof of Principle – Kinase Inhibitors • Test: promiscuous kinase inhibitors with a known spectrum of targets: staurosporine and GSK318257 • K562 cell extract treated with staurosporine or vehicle • Shallow slope -> less reproducibility • Because deviations in MS analysis will have bigger effects on Tm • 92% of detected proteins yielded sufficiently steep slopes Proof of Principle – Kinase Inhibitors • Test: promiscuous kinase inhibitors with a known spectrum of targets: staurosporine and GSK318257 • K562 cell extract treated with staurosporine or vehicle • Most affected proteins show positive shift • Some: destabilization (protein kinase C family) Proof of Principle – Kinase Inhibitors • Test: promiscuous kinase inhibitors with a known spectrum of targets: staurosporine and GSK318257 • K562 cell extract treated with staurosporine or vehicle • Comparison to previous study using chemoproteomics “kinobeads” profiling Identified kinases: Kinobeads: 229 Overlap: 66 Proof of Principle – Kinase Inhibitor Promiscuity • Thermal shifts identified for proteins other than kinases • coproporphyrinogen- III oxidase and ferrochelatase (FECH) (2/8 enzymes in heme biosynthesis pathway) Proof of Principle – Kinase Inhibitor Promiscuity • Thermal shifts identified for proteins other than kinases • coproporphyrinogen- III oxidase and ferrochelatase (FECH) (2/8 enzymes in heme biosynthesis pathway) • Regulatory components of other kinase complexes -> eg protein kinase A (PKA) complex • Inhibition by staurosporine appeared to stabilize the catalytic subunit but destabilize the regulatory subunit. Subunit: catalytic regulatory catalytic regulatory cAMP causes dissociation of reg from cat subunit Suggests: Catalytic subunit released from regulatory Correlation Between Ligand Affinity and Thermal Shift (R2 = 0.9) Comparing Staurosporine with GSK3182571: structurally divergent promiscuous kinase inhibitor Identified 13 common targets For these targets: both compounds showed similar Tm shifts -> suggesting that for a given protein, the Tm shift at saturating ligand concentrations is dependent on the intrinsic affinity of the ligand. affinity below 1 mM and a less than 10-fold difference in affinity. Isothermal Dose-Response (ITDR) • ITDR is generated at a defined temperature , over a range of compound concentrations • Affinity data for GSK3182571 • Good reproducibility, comparison with kinobeads -> good aggreement Testing Known Drugs For Affinity to Targets • Deficiency in FECH and results in high tissue levels of protoporphyrins • Vemurafenib (melanoma drug) often causes photosensitivity and increased levels of protoporphyrins -> • Profiling identifies known target BRAF and also FECH • Alectinib -> anaplastic lymphoma kinase (ALK) inhibitor (non-small lung cancer) -> also causes photosensitivity • FECH affected most potently by alectinib, then vemurafenib and not by crizotinib Demonstrates: TPP can serve as a standalone technique for obtaining quantitative affinity data for target- ligand interactions in a cell-based setting. Identifying Induced Tm Shifts in Downstream Proteins • Hypothesis: • Cell extract -> no downsream effects can be detected • Intact cells -> active signalling takes place -> downstream signalling could be detected Destabilized in intact cells No impact in cell extract Drug Concentration Dependance of Effektors • ITDR profiles at 3 temperatures • Half maximal reponse of CRKL marker was between 1.5 and 3.2 nM (agrees with known potency for inhibiting cell growth) CRKL , intact cells Destabilization of Effector with increase in desatinib concentration Positives and Drawbacks – TPP Positive Drawback • Identification of off-targets • False negative: Some ligands don’t provide a tm shift, therefore identification of all targets is not • Identification of direct targets and their effectors guaranteed (dasatinib –BCR, Savitski et al) • Dose dependency of targets and their effectors • Thermal profiling will miss proteins owing to • Possible identification of post-translational insufficient abundance and/or solubility or the modifications, fusion proteins, splice variants absence of a significant ligand effect. (typically undersamples in MS-based proteomics) • Application not developed in adherent cell systems • bound ligands, cofactors, metabolites, drugs yet • Can provide general view of proteomic state or • No detection of membrane proteins proteotype • Avoiding the design